Functional Diversity of Resilin in Arthropoda

Home , Cannula (grasshopper), Resilin, Vagrant Darter, Zyxomma

Functional diversity of resilin in Arthropoda

Jan Michels*, Esther Appel and Stanislav N. Gorb

Review Open Access

Address: Beilstein J. Nanotechnol. 2016, 7, 1241–1259. Department of Functional Morphology and Biomechanics, Institute of doi:10.3762/bjnano.7.115 Zoology, Christian-Albrechts-Universität zu Kiel, Am Botanischen Garten 1–9, D-24118 Kiel, Germany Received: 18 April 2016 Accepted: 15 July 2016 Email: Published: 01 September 2016 Jan Michels* - [email protected] This article is part of the Thematic Series "Biological and biomimetic * Corresponding author materials and surfaces".

Keywords: Associate Editor: K. Koch biological materials; biomechanics; composites; elastomeric proteins; functional morphology © 2016 Michels et al.; licensee Beilstein-Institut. License and terms: see end of document.

Abstract Resilin is an elastomeric protein typically occurring in exoskeletons of arthropods. It is composed of randomly orientated coiled polypeptide chains that are covalently cross-linked together at regular intervals by the two unusual amino acids dityrosine and trityrosine forming a stable network with a high degree of flexibility and mobility. As a result of its molecular prerequisites, resilin features exceptional rubber-like properties including a relatively low stiffness, a rather pronounced long-range deformability and a nearly perfect elastic recovery. Within the exoskeleton structures, resilin commonly forms composites together with other proteins and/or chitin fibres. In the last decades, numerous exoskeleton structures with large proportions of resilin and various resilin functions have been described. Today, resilin is known to be responsible for the generation of deformability and flexibility in membrane and joint systems, the storage of elastic energy in jumping and catapulting systems, the enhancement of adaptability to uneven surfaces in attachment and prey catching systems, the reduction of fatigue and damage in reproductive, folding and feeding systems and the sealing of wounds in a traumatic reproductive system. In addition, resilin is present in many compound eye lenses and is suggested to be a very suitable material for optical elements because of its transparency and amorphousness. The evolution of this remarkable functional diversity can be assumed to have only been possible because resilin exhibits a unique combination of different outstanding properties.

Review Resilin – the pliant protein Elastomeric proteins occur in a large range of organisms and bi- abductin of bivalve molluscs and gluten of wheat [1]. Besides ological structures, and the spectrum of their biological func- spider silk proteins, resilin is certainly the best-known among tions is very broad [1]. They feature a great diversity including the elastomeric proteins existing in arthropods. The first de- well-known examples such as elastin, titin and fibrillin present scription of resilin, which has often been called rubber-like pro- in vertebrate muscles and connective tissues, byssus and tein, was based on analyses of three different insect exoskel-

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eton elements: the wing hinge and the prealar arm of the desert the fully cross-linked protein is called resilin, whereas the not locust (Schistocerca gregaria) (also described for the migratory yet cross-linked or not fully cross-linked protein is called pro- locust (Locusta migratoria), Figure 1A,B) and the so-called resilin [6]. Within hydrolysates of resilin, glycine constitutes the elastic tendon of the pleuro-subalar muscles in dragonflies of largest proportion (30–40%) of the total residues [7,8]. Such the genus Aeshna [2]. Additional insights into the character- hydrolysates also feature the two unusual amino acids dityr- istics of resilin that had been gained shortly after this descrip- osine and trityrosine, which were identified to form the cross- tion [3,4] resulted in a comprehensive compilation of the then links between the polypeptide chains [9]. existing knowledge of resilin properties [5]. Resilin consists of a network of randomly orientated coiled polypeptide chains that The mechanical properties of resilin strongly depend on the have a high degree of flexibility and mobility and are linked degree of hydration because resilin is plasticised by water [5]. together at regular intervals by stable covalent cross-links. Only When resilin is completely hydrated, it behaves close to a

Figure 1: Occurrence of resilin in insects and crustaceans. Confocal laser scanning micrographs showing a wing hinge (A) and a prealar arm (B) of the migratory locust (Locusta migratoria), a wing vein joint of the common darter (Sympetrum striolatum) (C) and the ventral side of a female copepod of the species Temora longicornis (D). (A–C) Overlays of four different autofluorescences exhibited by the exoskeleton. Blue colours indicate large proportions of resilin, while green structures consist mainly of non- or weakly-sclerotised chitinous material, and red structures are composed of relatively strongly sclerotised chitinous structures. (D) Blue = autofluorescence of resilin, red = Congo red fluorescence of stained chitinous exoskeleton parts, green = mixture of autofluorescence and Congo red fluorescence of stained chitinous exoskeleton parts. (A, C, D) Maximum intensity projections. (B) Optical section. Scale bars = 100 µm (A, B), 20 µm (C), 200 µm (D). (A–D) Adapted with permission from [10], copyright 2011 John Wiley and Sons.

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perfect rubber [3,4,11]. Due to its molecular prerequisites, In order to allow a classification of exoskeleton structures as resilin then exhibits a near-perfect resilience of up to 92–97% resilin-containing exoskeleton according to the definition of and a fatigue limit of over 300 million cycles [12]. With respect Andersen and Weis-Fogh [5], these structures must conform to to resilience, resilin is unmatched by any other elastomeric pro- the wing hinge, the prealar arm and the elastic tendon tein and the best synthetic rubbers such as unfilled poly- mentioned above with respect to their properties, which can be butadiene [13,14]. Fully hydrated resilin has a rather low stiff- tested with a number of methods [5]. Resilin is colourless, ness. In the elastic tendons of dragonflies and locust ligaments transparent and amorphous. Accordingly, structures with very mentioned above, it was found to have a Young’s modulus of large resilin proportions can be easily distinguished from struc- 0.6–0.7 MPa and 0.9 MPa, respectively [11]. In addition, fully tures with relatively large proportions of chitin that are typical- hydrated resilin can be stretched to more than three times its ly pigmented and only slightly transparent or sometimes, when original length and compressed to one third of its original the sclerotisation is very pronounced, not transparent at all. length, and when the tensile and compressive forces are re- When immersed in aqueous media and in many anhydrous leased, resilin goes back to its initial state without having any hydrophilic liquids, resilin exhibits an isotropic swelling, which residual deformations [3,4,13]. is reversible and depends on the pH. (It is least pronounced at pH values of about 4.) In its hydrated state, resilin is swollen Until today, resilin has been found to exist mainly in insect and features its typical rubbery nature, long-range deformab- exoskeleton structures where this protein has a number of ility and complete elastic recovery. Furthermore, if hydrated different functions, which include (1) the storage of elastic resilin is tensioned, it will become birefringent, and the birefrin- energy in jumping systems [15-20], (2) the reduction of fatigue gence will be positive in the direction of the extension. When in folding wings of beetles and dermapterans [21,22], (3) resilin is completely dried, it loses its rubber-like character- the enhancement of the adaptability of attachment pads to istics and becomes relatively hard and brittle. Proteolytic en- uneven surfaces [23] and (4) the generation of flexibility of zymes such as pepsin or trypsin can be applied to test for the wing vein joints in dragonflies and damselflies [24-26]. Resilin presence and distribution of resilin, because resilin is known to has also been reported to be present in the exoskeletons of other be digested by such enzymes. Resilin has been shown to be arthropod taxa such as crustaceans [10,27-30] (Figure 1D), stained by single conventional dyes. Chemical reactions with scorpions [31] and centipedes [32]. In addition, resilin-like pro- the Masson and Mallory dyes were mentioned to stain resilin teins that contain dityrosine and trityrosine are known to exist in red. Staining of resilin with aqueous solutions of methylene several non-arthropod taxa including monogeneans [33,34], blue and toluidine blue is a common method and can provide nematodes [35], mussels [36] and sea urchins [37] indicating good information about the presence and distribution of resilin. that resilin likely originated much earlier in the evolution of When resilin is stained with one of these two dyes, it does not invertebrates than previously assumed. show metachromasia. Among the amino acids that form resilin, dityrosine and trityrosine exhibit a relatively pronounced auto- The properties of resilin-containing exoskeletons can strongly fluorescence. This autofluorescence is present in natural resilin- differ between structures and organisms. The reason is that in containing structures and in isolated resilin (both before and biological structures resilin seems to be rarely present in pure or after boiling in water) and in resilin hydrolysates. In neutral and nearly pure form but is known to commonly exist together with alkaline solutions, its excitation and emission maxima are at other proteins and/or chitin fibres in resilin-containing compos- about 320 nm and 415 nm, respectively [38,39]. The excitation ites, which exhibit a mixture of the properties of the single com- spectrum of the resilin autofluorescence differs with changing ponents. In such composites, the resilin properties can even be pH conditions. In acid solutions, the excitation maximum is ‘overlain’ by the properties of the other components making an shifted considerably to about 285 nm, and the upper edge of the identification of the presence of resilin in the respective struc- excitation peak is at about 330–340 nm [38,39]. The pH-in- tures with the criteria of Andersen and Weis-Fogh [5] very duced changes of the excitation properties are reversible and difficult. In addition, certain exoskeleton structures feature only take place rapidly [40]. some of the typical characteristics of resilin-containing material but lack the others. It is then often not possible to determine The described resilin identification and visualization methods whether these structures contain resilin or other proteins resem- are not absolutely specific. Therefore, it is strongly advisable to bling resilin. In such cases, it is conceivable that the respective apply not only one single method but a combination of several exoskeleton material consists either of a protein with properties different ones to increase the reliability of the identification and that are similar to those of resilin or of a mixture of resilin and detection of resilin. In recent years, an antibody to a recom- other proteins. For exoskeleton structures with such properties, binant Drosophila melanogaster pro-resilin (rec1-resilin) was the term ‘transitional cuticle’ was established [5]. developed and has been shown to be cross-reactive and to label

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resilin in different insects [12,13,41,42]. Until today, this modulus of such setae was measured along the longitudinal axis immunohistochemical method has been tested for only a small of the setae (Figure 2C). The measurements revealed that the number of insects and only within the studies mentioned above. Young’s modulus of the material in the most distal section of If it proves efficient in tests with a larger number of arthropod each seta is relatively low (1.2 ± 0.3 MPa), whereas it is consid- species, it will represent the first reliable method that specific- erably higher at the setal base (6.8 ± 1.2 GPa). The differences ally identifies resilin. in the Young’s modulus between different regions correlate with the resilin proportion observed in the seta material [48]. The development and improvement of methods applying tech- When the setae are dehydrated, the Young’s modulus of the niques such as micromechanical testing, atomic force microsco- setal tip material strongly increases from 1.2 to 7.2 GPa, and it py and confocal laser scanning microscopy (CLSM) have facil- exhibits no statistically significant differences along the com- itated detailed studies of the distribution, composition and me- plete setae [48], which is in accordance with the relationship be- chanical properties of resilin-containing exoskeleton structures tween the material properties and the hydration status of resilin in diverse organisms and at different levels of their organisa- mentioned above. Besides the differences in the Young’s tion. One of these methods utilises a combination of different modulus, the mechanical behaviour of the respective materials autofluorescences. In addition to resilin, other arthropod shows the pronounced differences in the material composition exoskeleton materials also exhibit autofluorescences, which can between the tips and the bases of fresh adhesive tarsal setae. be efficiently visualized with fluorescence microscopy. This While the material of the tip features only elastic deformation, allows the production of overlays consisting of different micro- both elastic and, to some extent, plastic deformation are ob- graphs that show different autofluorescences. Such overlays served in the material of the base [48]. This means that the nicely exhibit differences in the autofluorescence composition, purely elastic response of the tip is due to the presence of which are good indications for differences in the material com- resilin, whereas the partially plastic deformation at the base is position and clearly reveal structures with relatively large resilin mainly due to the presence of stiffer tanned exoskeleton. It is proportions within the analysed specimens [21,23,43]. Howev- very likely that effects similar to those observed in beetle er, when analysing arthropod exoskeleton structures for the adhesive tarsal setae exist in other exoskeleton structures with presence of the autofluorescence of resilin, one has to bear in comparable gradients of the resilin proportion. mind that some other compounds present in organisms exhibit autofluorescences whose properties are similar to those of the Occurrence and functions of resilin in differ- resilin autofluorescence, with excitation maxima in the UV ent arthropod exoskeleton systems range and emission maxima in the violet and blue ranges of the Resilin is known from numerous arthropod exoskeletons where light spectrum [44-47]. it is present in diverse structures and allows manifold functions, which in most cases are based on its very pronounced elasticity If the autofluorescences are visualized by means of CLSM, the and its ability to completely recover after deformation. For ex- results will be very detailed and precise [10]. With a recently ample, resilin plays an important role in flight systems of described method, four different autofluorescences are excited insects, in particular in insects that use a wing beat with a low and detected separately, and certain colours are allocated to frequency (10–50 Hz) (see below). Resilin-containing exoskel- each of the four visualized fluorescence signals. On the result- eton structures have been described for various mechanical ing overlays, exoskeleton structures with large proportions of systems including leg joints [40,50], vein joints and mem- resilin are blue, while green structures consist mainly of non- or branous areas of insect wings [21,22,24], the food-pump of weakly-sclerotised chitinous material, and red structures are reduviid bugs [51], tymbal sound production organs of cicadas composed of relatively strongly sclerotised chitinous structures [52,53] and moths [54], abdominal cuticle of honey ant workers (for details see [10]; Figure 1A–C). Many of the confocal laser [55] and termite queens [56], the fulcral arms of the poison scanning micrographs shown in this review were created using apparatus of ants [57] and the tendons of dragonfly flight this method. muscles and basal wing joints of locusts (as already mentioned above) [5]. In the following, some selected representative struc- Very often, gradients of the material composition with a consid- tures and systems with large proportions of resilin are high- erably changing proportion of resilin are present in arthropod lighted, and their functions are described. exoskeleton structures. Such resilin proportion gradients must also be reflected by gradients of the mechanical properties of Arthrodial membranes the respective resilin-containing composites. The material com- Arthrodial membranes are cuticle areas that are typically thin, position of adhesive tarsal setae of beetles (Figure 2B) repres- non-sclerotised and very flexible. Such membranes often are ents a good example for such gradients. Recently, the Young’s multifunctional units. The soft cuticles of caterpillars, for exam-

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Figure 2: Distribution, mechanical properties and functional significance of resilin in adhesive tarsal setae. (A, B) Ventral part of the second adhesive pad of a female seven-spot ladybird (Coccinella septempunctata), lateral view. (A) Scanning electron micrograph. (B) Confocal laser scanning micrograph (maximum intensity projection) showing an overlay of four different autofluorescences exhibited by the exoskeleton. Blue colour indicates the presence of large proportions of resilin. (C) Box-and-Whisker plots showing the median Young’s modulus of fresh adhesive tarsal setae obtained by atomic force microscopy nanoindentations (see the inset above the graph) along each seta. The borders of the boxes define the 25th and 75th percentiles, the median is indicated by a horizontal line, and the error bars define the 10th and 90th percentiles (n. s. = not significant, *** = highly significant). The background colours indicate the different seta sections. (D–F) Numerical model showing typical configurations of a filamentary structure (setal array) attached to a stiff support (black rectangle) in adhesive contact with a random fractal surface (blue region). Three types of fibres were tested: (D) stiff fibres with short elastic ends, (E) long elastic fibres connected to the base by short stiff roots and (F) stiff fibres with soft elastic segments near the base. The different stiffnesses of the segments are conditionally shown by circles with different colours. Stiff, medium and soft segments are marked by black, red and green circles, respectively. Scale bars = 25 µm (A, B). (A–C) Adapted with permission from [48], copyright 2013 Nature Publishing Group. (D–F) Adapted with permission from [49]. ple, have a combination of both a protective and a locomotory forms a spring-like (or joint-like) element (Figure 3A–C) that role, which is reflected in their ultrastructural architecture [58]. makes the pulvilli movable and thereby enables them to effi- The main functions of arthrodial membranes are to connect ciently adapt to the substrate. In general, joints in legs typically sclerotised exoskeleton elements and allow relative movement feature membranes, which often contain large proportions of of these elements and to extend whenever an increase in volume resilin and allow the relative movement of the joint elements of the body is necessary [59,60]. In addition, some membranes (Figure 3D). are armoured with miniature protuberances on their surfaces and have a defence function [61,62]. For insects, two different The neck membrane of dragonflies is another example. This types of membranes have previously been reported. The first flexible cuticle connects the neck sclerites and enables an exten- type is a highly extensible membrane found in the locust sive mobility of the head [69]. A recent study clearly revealed abdomen that can extend up to ten times its original length [63- that the neck membrane material of the broad-bodied chaser 65]. This cuticle is highly specialised in its protein composition (Libellula depressa) contains relatively large proportions of [66]. The second type is a folding laminated membrane that is resilin, while the neighbouring sclerites are mainly composed of less stretchable and has been found, for example, in the sclerotised chitinous material [10] (Figure 3E). Transmission abdomen of the tsetse fly Glossina morsitans [66] and in the electron microscopy showed that dragonfly neck membrane bug genus Rhodnius [67,68]. cuticle is rather homogenous and electron-lucent [69]. Mem- branous areas of insect cuticle nearly always exhibit a relative- Membranous cuticle often contains large proportions of resilin ly intensive autofluorescence similar to that of resilin. This sug- (Figure 3). Examples are membrane structures connecting claws gests that arthrodial membranes generally contain relatively and pulvilli to the terminal tarsomere [43,50]. In the pretarsus of large proportions of resilin. However, even very soft and flex- the drone fly (Eristalis tenax) (Insecta, Diptera, Syrphidae), for ible membranes such as the dragonfly neck membrane do not example, membranous cuticle with large proportions of resilin consist of pure resilin but rather represent resilin–chitin com-

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Figure 3: Resilin in arthrodial membranes of insects. (A, B) Pretarsus of the drone fly (Eristalis tenax), ventral view. (A) Overlay of a bright-field micrograph and a wide-field fluorescence micrograph showing the presence and distribution of resilin autofluorescence (blue). (B) Confocal laser scanning micrograph (maximum intensity projection) revealing large proportions of resilin (shown in blue) in the membranous structures between rigid sclerites of the pretarsus. (C) Pretarsus of the urban bluebottle blowfly (Calliphora vicina). Transmission electron micrograph showing an ultra-thin section through structures comparable to those shown in blue in B. (D) Lateral view of the joint between the tarsomeres 1 and 2 in the third leg of the seven- spot ladybird (Coccinella septempunctata). (E) Border between the neck membrane (right side) and a postcervical sclerite (left side) of the broad- bodied chaser (Libellula depressa). (F, G) Membranous cuticle in the neck area of the blue-tailed damselfly (Ischnura elegans). Transmission electron micrographs showing ultra-thin sections. EPI, epicuticle; EXO, exocuticle; FD, folds; VES, vesicles. Arrows: preferential orientation of chitin fibres. Scale bars = 100 µm (A), 50 µm (B), 2 µm (C), 20 µm (D, E), 1 µm (F, G). (A, B, D, E) Adapted with permission from [10], copyright 2011 John Wiley and Sons. (F, G) Adapted with permission from [69], copyright 2000 The Zoological Society of London. posites in which some reinforcement by chitin-bearing micro- vulnerata) (Cercopidae), the complete extension of the hind leg fibrils is clearly visible (Figure 3F,G). takes less than one millisecond [16] (Figure 4E). This suggests that, in addition to the muscle system, an elastic spring system Legged locomotion powers the jump. The application of fluorescence microscopy Mechanisms of fast leg movements with an acceleration that and histological staining revealed structures with large propor- can surpass the limitations of muscle contraction have been tions of resilin in the pleural area of the metathorax found in different insect groups including fleas [15,70], locusts (Figure 4A–D). These structures stretch dorso-ventrally across [71], beetles [72,73] and true bugs [16-18,74]. The respective the entire pleural area (Figure 4F) and are much larger than catapult-like devices have often evolved to enhance the acceler- comparable structures present in fleas (see below). Their dorsal ation in relatively short legs [75]. They usually contain specific and ventral parts are located close to the origin of the lateral types of joints that are typically supplemented with active portion of the power muscle and closely connected to the lateral power or latch muscles producing tractive force and trigger part of the coxa, respectively. The resilin-containing structures muscles that are responsible for releasing elastic energy from very likely participate in the extension of both coxa and specific energy storage devices (see below). trochanter by the release of energy that is stored by deflection or twisting of their bar-like shape. In Auchenorrhyncha and Sternorrhyncha, the jumps are per- formed by metathoracic muscles that are directly connected to Fleas possess two pads with large proportions of resilin in their the trochanter of the hind leg and responsible for the move- thorax [15]. These pads are located at the hindlegs and associat- ments of both trochanter and coxa [16,74] (Figure 4F). In the ed with the trochanteral depressor muscles that actuate the jumping cicada called black-and-red froghopper (Cercopis jumps. Before each jump, the pads are compressed, and then

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Figure 4: Resilin in the jumping system of the black-and-red froghopper (Cercopis vulnerata). (A–D) Cuticle supplemented with resilin in the pleural area of the metathorax. Horizontal sections through the pleural seam. (A) Paraffin-embedded cuticle stained after Cason. Bright-field micrograph. (B–D) Frozen sections of the structures shown in A. Wide-field fluorescence micrographs showing different autofluorescences exhibited by the exoskeleton. (B) Excitation: 512–546 nm; emission: 600–640 nm. (C) Excitation: 340–380 nm; emission: 420 nm. (D) Excitation: 710–775 nm; emission: 810–890 nm. cu, tanned cuticle; RES, structures containing resilin. (E) Sequence of single frames of a high-speed video recording of the cicada jump (ventral aspect). The numbers indicate the time scale in milliseconds. The approximate positions of femur, tibia and tarsus are indicated by white lines according to white marker dots on the leg. (F) Skeleton-muscle organisation of the cicada metathorax (medial aspect, right side). AP, apodeme of the trochanter extensor muscle M3; CX, coxa; FE, femur; M1, M2, M4, M6, M14, subcoxal muscles; M7, M9, M10, M11, M12, M13, M14, metathoracic muscles; M5, trigger muscle; M8a, M8b, trochanter flexor muscle; RES, resilin; TN, trochantine; TR, trochanter; white circles, condyli of the coxa and trochanter. The inset shows the position of the structures in the entire insect body. (A–F) Adapted with permission from [16], copyright 2004 Elsevier. their elastic recoils provide energy for the rapid trochanter the energy that is necessary to fulfil the large power demands of movements powering the jump. While fleas perform their jumps fast leg movements involved in actions such as jumping [17]. just with the hindlegs, snow fleas (Mecoptera, Boreidae) use Energy storage devices used for jumping in froghoppers (Hemi- their hind and middle leg pairs for jumping. They feature four ptera, Cercopidae) and planthoppers (Hemiptera, Issidae) and resilin-containing pads, one at each of the legs involved in for jumping and kicking in locusts were shown to be compos- jumping, and these pads are similar to those of fleas with ites of relatively hard and stiff chitinous structures and struc- respect to their locations at the legs and their function [19]. tures with large proportions of resilin [17,18,20]. It was suggested that a large proportion of the energy needed for jumping For the jumping mechanism of fleas it was proposed that the is stored within the hard and stiff chitinous structures, which whole energy required is stored in the resilin-containing pads (because of the stiffness of the material) likely requires only [15]. However, the results of recent estimations indicate that small amounts of bending and, therefore, only short muscle resilin alone often can provide only a rather small proportion of contractions [17,18,20]. The flexibility and elasticity of resilin

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are assumed to facilitate this mechanism by reducing the risk of membrane cells, and cuticular layers within wing veins fractures occurring within the stiffer material and by contribut- [2,5,21,22,77-79]. In these structures, resilin occurs either in the ing to a rapid and complete return of the distorted energy form of pure or nearly pure resilin (e.g., tendons) or mixed with storage devices to their original shapes [17,18,20]. It is conceiv- varying amounts of chitin fibres (e.g., prealar arm), which tend able that a large proportion of the energy storage devices to follow distinct directions or patterns and thereby influence existing in insects with fast leg movements, including those of the mechanical properties of the material [5,28]. All of these fleas, feature composite architectures comparable to those de- structures benefit to a more or less pronounced extent from the scribed above. presence of resilin due to its low stiffness, high resilience, large and reversible extensibility, long fatigue time and ability of The ability of resilin to store energy within jumping systems elastic energy storage and damping. was shown to be also involved in an energy absorption function of a specific structure, called buckling region, that is present in One of these flight system elements is a sausage-like swollen each tibia of locust hind legs [76]. The buckling region is locat- thoracic dragonfly tendon, which consists of virtually pure ed in an area where the bending moment during jumping and resilin and connects the pleuro-subalar muscle (which spans be- kicking is high. When a hindleg slips during jumping or misses tween the lower part of the pleuron and the subalar sclerite) to a target during kicking, this structure can buckle and thereby act the subalar sclerite, which in turn connects to the axillary scler- as a shock absorber by dissipating energy that would otherwise ites of the wing base [2] (Figure 5A,B). Together with the have to be absorbed by other suctures such as the leg joints. The coxoalar muscle, it is assumed to control wing twisting (i.e., buckling region exhibits parts with large proportions of resilin supination) during the upstroke by taking up wing movements that are assumed to contribute to the energy absorption and to and oscillating in length, while the attached pleuro-subalar the restoration of the original shape of the leg after buckling muscle contracts slowly and tonically and keeps the tendon at a [76]. certain length and tension [5,80,81]. This is especially important for hovering and other refined flight manoeuvres [9]. It Flight systems: folds, tendons and microjoints might also play a role in controlling excess wing motions during Resilin has already been found in various elements of insect turbulent flows [81]. In the tendon, large reversible extensib- flight apparatus, including tendons connecting muscles to ility (e.g., over 250% in the forewing of the widow skimmer pleural sclerites, wing hinge ligaments connecting the wings to (Libellula luctuosa)) and a long fatigue time are of key import- the thoracic wall, prealar arms connecting pleural sclerites to ance for the functioning of this structure. the mesotergum, wing vein micro-joints connecting cross veins to longitudinal veins, regions of the wing membrane estab- The wing hinge ligament of the forewing of locusts is located lishing a connection to the wing veins or defined patches within between the (meso-)pleural wing process and the second axil-

Figure 5: Resilin in flight systems of dragonflies. (A) Stereomicrograph depicting a resilin-bearing tendon (rt) of a pleuro-subalar muscle (ps) of the southern hawker (Aeshna cyanea). (B) Overlay of a bright-field micrograph and a wide-field fluorescence micrograph showing autofluorescence (blue) exhibited by resilin in a tendon of the genus Zyxomma. (C) Flexible wing vein joints (fj) joining cross veins (cv) to a longitudinal vein (lv) in a wing of the vagrant darter (Sympetrum vulgatum). (D) Cross section through a wing vein of S. vulgatum, revealing sclerotised exocuticle and resilin-bearing endocuticle. (C, D) Confocal laser scanning micrographs (maximum intensity projections) showing overlays of different autofluorescences exhibited by the exoskeletons. Structures with large proportions of resilin are shown in blue. Scale bars = 1 mm (A), 100 µm (B), 40 µm (C), 10 µm (D). (B) Adapted with permission from [13], copyright 2005 Nature Publishing Group. (D) Adapted with permission from [79], copyright 2015 John Wiley and Sons.

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lary wing sclerite [5]. In Odonata, Dictyoptera and Orthoptera, contraction, the prealar arm is deformed and can be assumed to wing hinge ligaments exist in the form of thick, rubber-like play a role in elastic energy storage. pads (Figure 1A) and, as reported for locust structures, consist of a rather tough, mainly chitinous ventral part and a soft dorsal Cross veins in wings of dragonflies and damselflies were shown part, which can be divided in a region of pure resilin and a to form either stiff, inflexibly fused joints or flexible, resilin- region containing both chitin lamellae and resilin [5]. The bearing joints to the adjacent longitudinal veins [24-26,78,86] results of several studies suggest that wing hinge ligaments take (Figure 5C). The distribution pattern of different wing vein joint up compressive as well as tensile forces and can contribute to types on the dorsal and ventral wing sides in various species is (1) the storage of kinetic energy at maximum wing deflection, quite diverse, but was found to follow phylogenetic trends prob- for example during the upstroke when the wing hinge ligament ably related to wing morphology and flight behaviour [26,78]. is stretched, and (2) wing acceleration during the downstroke by In general, flexible wing vein joints, together with the overall elastic recoil [5,28,82]. In other insects, such as Lepidoptera, corrugated design of odonate wings, are assumed to feature a some Coleoptera and some Hymenoptera, these ligaments are larger angular displacement than fused vein joints and, as a tough and inextensible, and elastic energy storage is likely pro- result, to provide the wing with increased chord-wise flexibility, vided by the rigid thoracic cuticle and the flight muscle itself which promotes passive wing deformations such as camber-for- [5,28]. Due to the fact that resilin has mainly been found in the mation during the downstroke, and, thereby, to improve the flight apparatus of insects flying with synchronous flight aerodynamic and mechanical performance of the wing muscles at low wing beat frequencies of less than 50 Hz and [24,26,78,86,87]. Moreover, resilin is important for reducing with inertial forces being larger than aerodynamic forces, it is stress concentrations in vein joints [87]. Resilin is not only assumed that its resilience might be too small at high frequen- present in wing vein joints but also in the wing membrane cies [5,11,28,83]. However, there is still some controversy directly abutting on wing veins and internal cuticle layers of about the frequency-dependent behaviour of resilin and wing veins (i.e., the endocuticle) [78,79] (Figure 5D). A flex- chitin–resilin composites and its function in the wing hinge ible suspension of the wing membrane is suggested to allow ligaments of insects with high wing beat frequencies [5]. For larger strain and thereby to help preventing its tear-off from the example, some small wing hinge ligaments have been found be- wing veins [79]. Furthermore, the stiffness gradient in wing tween different sclerites in the genera Calliphora, Bombus, Apis veins, generated by a stiff, sclerotised outer layer (exocuticle) and Oryctes [5,84]. So far, only a few studies have investigated and a soft, compliant, resilin-bearing inner layer (endocuticle) is the decrease in resilience with increasing frequencies in the assumed to reduce the overall vein stiffness and to improve the dragonfly tendon, locust prealar arm and cockroach tibia-tarsal damping properties of the vein as well as to delay Brazier oval- joint resilin [5,11,81,83,85]. Whether the partly pronounced isation and to enhance the load-bearing capacity under large de- differences in the decrease rate of resilience between different formations [79,88]. frequency ranges are due to different measurement techniques or are actually due to differences in the material composition, By artificially stiffening single flexible, resilin-bearing vein still needs to be elucidated. joints in bumblebee wings through the application of micro- splints (extra-fine polyester glitter glued with cyanoacrylate), it The prealar arm is located at the front edge of the mesotergum was experimentally shown that even a single resilin-bearing and establishes a connection to the first basalar sclerite of the joint plays an important role in overall wing flexibility and pleural thoracic wall via a tough, flexible ligament [5]. The vertical aerodynamic force production [89]. Ma et al. [90] found basalar sclerite in turn is connected to the humeral angle of the comparable resilin joints (e.g., the 1 m-cu joint) in wings of anterior part of the wing base. The prealar arm consists of western honey bees (Apis mellifera) and assumed that they around 23% chitin and 77% resilin and is structured by altern- might play an analogous role in increasing the chordwise wing ating layers of resilin and chitin fibrils, with the fibrils flexibility. Based on the distribution of resilin patches, wing continuing into the dark, sclerotised cuticle at its base [5] veins, the occurrence of a flexible hook-mediated (Figure 1B). Due to the directional arrangement of chitin fibrils, forewing–hindwing connection and observed wing deforma- the mechanical behaviour of the prealar arm is assumed to be tions, they further suggested the existence of five flexion lines dominated by the mechanical properties of the chitin fibrils in one forewing–hindwing entity and assumed that these proba- during stretching and by the properties of resilin during bending bly increase the cordwise flexibility and support camber forma- and compression [5,11]. In contrast to the subalar muscle, tion. In addition, Mountcastle and Combes [91] demonstrated which is involved in wing supination, the contraction of the that a resilin-bearing joint at the leading edge (the costal break) basalar muscle causes wing pronation through the connection to in the wings of wasps plays a major role in mitigating wing the humeral angle via the basalar sclerite. During muscle wear by flexion along this joint when the wings hit an obstacle.

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This mechanism is especially important for wings with wing tribution of the maximum spanwise bending stress at the begin- veins extending all the way to the tip because such a design ning of each stroke cycle and suggests that the resilin patches endows a wing with more spanwise rigidity than, for example, reduce the risk of breaking near the wing hinge due to a de- bumblebee wings that lack veins at the wing tip [91]. crease in peak stress in the rigid wing parts [77].

The occurrence of resilin in several broadened vein patches as Attachment systems well as in membranous folding lines was described for fan-like The contact formation of insect adhesive pads on substrates dermapteran hind wings [22,92]. These structures help folding depends on the ability of the pads to adapt to the surface topog- the wing into a wing package being ten times smaller than the raphy. In this context, specific micro- and nanostructures can unfolded wing. This package can then be hidden under the short enhance the quality of the contact [97-101]. In the case of sclerotised forewings. The four-fold wing folding can be attachment on rough substrates, multiple contacts, being formed achieved without musculature activity and is assumed to be by some adhesive systems, provide great advantages [102]. The driven by elastic recoil of the anisotropically distributed resilin formation of multiple contacts, which contribute to an increase on either the ventral or the dorsal sides of broadened vein of the overall length of the total peeling line, is facilitated by a patches in intercalary and radiating veins, supported by the hierarchical organisation of the attachment structures [103]. It resilin-bearing radiating folds that influence the folding direc- was shown that the combination of thin tape-like contact tips of tion [22]. Unfolding of the hind wings is achieved either by hairs (setae) and applied shear force lead to the formation of a wiping movements of the cerci (e.g., in the European earwig maximal real contact area without slippage within the contact (Forficula auricularia) and the lesser earwig (Labia minor)) or [104]. This indicates that material flexibility is very important by wing flapping (e.g., in the earwigs Timomenus lugens, which for the contact formation of adhesive pads. With a minimal has very long cerci, and Auchenomus sp.) [22,93]. Both normal load, flexible materials can create a large contact area unfolding mechanisms are supported by several wing stiffening between the attachment structures and the substrate. However, mechanisms such as the mid-wing mechanism and the claval elongated structures that are too flexible have a low mechanical flexion line, which keep the wing unfolded in all species exam- stability [105]. For example, if insect setae are too soft, they can ined [22,93]. These mechanisms were found to play an impor- buckle and collapse, and so-called clusterisation (or condensa- tant role both in the static unfolded state of the wing and during tion) can take place [106,107]. As a result of this, the func- flapping flight, in which they help to inhibit an unfavorable tional advantages achieved through multiple adhesive contacts folding of the wing [92]. Furthermore, the flexible resilin-bear- can be strongly reduced [103]. Accordingly, the composition ing folding lines were found to not only serve wing folding but and the properties of the material of insect adhesive setae also act as flexion lines at which the wing flexes during flight, represent an optimisation problem. There is evidence that thereby supporting the generation of an aerodynamically during the evolution gradients of the thickness and the mechani- favourable cambered wing profile [92,94]. cal properties of the setae have developed as a solution of this problem. The presence of thickness gradients, revealed by scan- In beetle wings, resilin was found to occur at the marginal joint, ning electron microscopy, is well-known for various insect between veins that separate during folding, and along flexion adhesive setae [97]. Recently, a gradient of the material compo- lines in membranous areas, leading to the hypothesis that elastic sition, present on the level of each single adhesive tarsal seta, energy storage by resilin can support wing unfolding also in was shown to exist in the seven-spot ladybird (Coccinella beetle wings [21]. However, this can, if at all, only be a septempunctata) [48] (Figure 2A–C). The material of the setal supportive role because wing unfolding in beetles was stated to tip contains large proportions of resilin, while the base of the be mainly achieved by scissor-like movements of the RA and seta consists mainly of sclerotised chitinous material. Between MP1+2 veins via contraction of the Musculus pleura alaris and the tip and the base, a pronounced material composition the basalar muscles, which is possibly supported by hydraulic gradient was revealed by CLSM. This gradient is reflected by a hemolymph pressure [95,96]. Like in dermapteran wings, in pronounced gradient of the material properties: the setal tip is beetle wings resilin most probably delays material fatigue in rather soft, whereas the setal base is relatively stiff. Both gradi- highly stressed wing regions and might further play a role in ents were hypothesised to represent an evolutionary optimisa- wing deformation during flight [22]. tion that increases the attachment performance of the adhesive pads when they attach to rough surfaces due to an efficient In wings of the urban bluebottle blowfly (Calliphora vicina), adaptation of the soft and flexible setal tips to the substrate and resilin is mainly present in the proximal part of the wing, pre- a simultaneous prevention of setal clusterisation by means of dominantly in the form of resilin-bearing patches between veins the stiffer setal bases [48]. Since this hypothesis is difficult to [77]. The occurence of resilin coincides with the proximal dis- test experimentally using biological specimens, it was tested

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using numerical simulations [49] (Figure 2D–F). The results in- gradients revealed in smooth adhesive pads of locusts and bush dicate that setae with long soft tips and rigid bases exhibit a crickets differ from those existing in the adhesive tarsal setae strong adhesion but also a pronounced clusterisation described above. The smooth pads contain a relatively soft core, (Figure 2E). Setae with rigid tips and soft bases have a low which is covered by a stiffer layer. Accordingly, the direction of adhesion and a pronounced clusterisation (Figure 2F). Only the material gradient is opposite to that in the adhesive tarsal setae with short soft tips and rigid bases feature optimal adhe- setae, which can be well explained by the different pad architec- sion properties and simultaneously a minimum of clusterisation ture. Smooth pads feature branching fibres (rods) that form (Figure 2D), which confirms the hypothesis. Tarsal liquids pro- foam-like structures. The spaces between the solid structures duced by beetles are assumed to contribute to the adhesion effi- are filled with fluid. Due to this construction principle, the pads ciency of adhesive pads in the form of capillary interactions and are kept in shape. The fibres are terminated by a relatively stiff cleaning effects. With regard to the resilin-dominated setal tips, superficial layer that keeps the positions of the relatively long an additional function is conceivable. As described above, and thin fibres (and thereby the distance between the fibre tips) resilin is only soft and flexible when it is hydrated. According- constant [23,108]. This pad architecture was studied in detail in ly, to keep the contribution of the large resilin proportions in the two orthopteran species, the great green bush-cricket (Tetti- setal tips to the attachment performance of the adhesive pads on gonia viridissima) (Ensifera) and the migratory locust (Locusta a high level, the hydration of the resilin must be maintained. It migratoria) (Caelifera), whose adhesive pads generally have a is imaginable that this is achieved by slowly evaporating tarsal similar structural organisation [23] (Figure 6A–D). Both pads liquids covering the setae and thereby keeping the resilin in the possess a flexible resilin-containing exocuticle with fibrils that setal tips hydrated [48]. are fused into relatively large rods oriented in an angle to the surface. However, slight differences in the pad architecture The presence of material gradients has also been demonstrated exist. Adhesive pads of L. migratoria feature a clearly thicker for smooth attachment devices of insects [23]. Interestingly, the superficial layer as well as a higher density of rods than those of

Figure 6: Material structure and properties of orthopteran adhesive pads (euplantulae). (A, B) Great green bush-cricket (Tettigonia viridissima). (C, D) Migratory locust (Locusta migratoria). (A, C) Scanning electron micrographs showing frozen, fractured, substituted, dehydrated and critical point dried pads. (B, D) Wide-field fluorescence micrographs showing frozen-cut pads. Structures with large proportions of resilin are violet/blue. sl, superficial layer; bl, layer of branching rods; rl, layer of primary rods. (E) Desiccation dynamics of tarsi and pieces of tibiae cut off the body in both species. (F) Effective Young's modulus of attachment pads of both species determined by means of indentation with a spherical tip radius of 250 µm. (G) Work of adhesion of attachment pads of both species measured by means of indentation with a sphere with a radius of 32 µm. In F and G, the ends of the boxes define the 25th and 75th percentiles, the lines indicate the medians, and the error bars define the 10th and 90th percentiles. (A–G) Adapted with permission from [23], copyright 2006 Springer.

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T. viridissima (Figure 6A–D). In addition, indentation experi- spirally coiled when it is in its resting position [113]. For the ments revealed a higher effective Young’s modulus and a lower uptake of food, hemolymph is pumped into the proboscis result- work of adhesion for L. migratoria pads (Figure 6F,G). The ing in the generation of hydrostatic pressure that completely lower adhesive properties of L. migratoria pads can be ex- uncoils the proboscis [113-115] and strongly changes the shape plained by the larger thickness of the relatively stiff superficial of certain proboscis elements. During this process, dorsal parts layer, which likely reduces the adaptability of the pad to the of the proboscis are compressed. These parts contain relatively substrate much more than the relatively thin superficial layer of large proportions of resilin and act as springs that cause the the T. viridissima adhesive pad. The superficial layer is recoiling of the proboscis when the hydrostatic pressure is assumed to also protect the pad from desiccation as indicated by removed [115]. experiments showing that cut-off adhesive pads of T. viridissima (with the relatively thin superficial layer) lose water A remarkable resilin-containing adhesive prey-capture device, much faster than those of L. migratoria (Figure 6E). which is formed by the elongated labium, exists in rove beetles Consequently, the material gradient provides a combination of of the genus Stenus (Staphylinidae). This prey-capture appar- conformability to the surface roughness of the substrate (The atus can be protruded towards a prey within a few milliseconds. compliant material of the pad contributes to the efficient con- When sticky pads (modified paraglossae), which are located at tact formation with the substrate.) and resistance to the dry the distal end of the prementum, adhere to the prey, the labium environment. Such pad architectures likely depend on the is withdrawn immediately, and thereby the prey is transported preferred environment of each species and are the result of to the mouth region of the beetle where it can be seized with the trade-offs between different factors such as evaporation rate, mandibles [116-118]. The sticky pads feature a surface that is stiffness, stability and adhesion. subdivided into numerous terminally branched outgrowths. During the prey capture, these surface structures are completely Mouthparts covered by an adhesive secretion that is produced in special The first mouthpart-related structures containing resilin were glands located in the head capsule and makes the sticky pads a already mentioned shortly after the description of resilin. In the hairy, hierarchically structured and wet adhesive system. Simi- respective studies, resilin was found in the salivary and feeding lar to the insect tarsal adhesive pads mentioned above, softness pumps of assassin bugs [109] (cited in [110]), [111]. Later, the and compliance of the pad cuticle contribute to the generation findings were confirmed and complemented by additional of strong adhesive forces by the pads. The cuticle material of information about the resilin distribution [51]. In these pumps, certain parts of the sticky pads contains large proportions of which enable the bugs to suck relatively large amounts of blood resilin providing flexibility and elasticity and enabling the pads in a short time period and to inject proteolytic enzymes into to efficiently adapt to the surface of the prey items [118]. prey or assaulters or to spit on the latter, the resilin-containing structures function as elastic spring antagonists to muscles. A Copepods are tiny crustaceans that inhabit nearly all aquatic similar function was described for resilin-containing structures habitats worldwide and are particularly abundant in the marine present in the maxillipeds of decapod crustaceans [112]. The water column where they contribute large proportions of the movements of the flagella of these mouthparts influence the zooplankton [119,120]. The diet of many of the marine plank- water flow through the gills as well as over chemoreceptors lo- tonic species comprises relatively large fractions of diatoms cated on the head, and thereby they importantly contribute to (i.e., unicellular algae with silica-containing shells called frust- active chemoreception and to signalling by distributing urine ules). Copepods use the gnathobases of their mandibles to grab odours. Each of the flagella is abducted by the contraction of a and mince food particles. To be able to efficiently digest the single muscle. Due to this abduction, a structure that contains diatom cells, the copepods must crack the frustules before the relatively large resilin proportions and is located in the joint be- ingestion of the cells. The gnathobases possess tooth-like struc- tween the flagellum und the exopodite of the maxilliped is bent. tures (called teeth in the following) at their distal ends [121]. In After relaxation of the muscle, this elastic structure recovers its copepod species feeding on large amounts of diatoms, these original shape and moves the flagellum back to its resting posi- teeth are rather compact and consist of complex composites that tion. combine diverse structures and materials with a wide range of properties. Recently, the morphology and material composition In general, due to its very pronounced elasticity and fatigue of the gnathobases of two copepod species have been analysed resistance, resilin appears to be a very suitable material for and described in great detail [29,30]. The gnathobases of the exoskeleton structures that are typically intensively deformed calanoid copepod Centropages hamatus feature two larger and for a rather large number of times during the lifetime of the relatively compact teeth (Figure 7A–E). Each of these teeth pos- organisms. A butterfly proboscis, for example, is tightly and sesses a chitinous socket, which is covered by a cap-like struc-

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Figure 7: Morphology and material composition of mandibular gnathobases of copepods. Confocal laser scanning micrographs showing gnathobase structures of (A–E) a female of the copepod species Centropages hamatus and (F, G) a female of the copepod species Rhincalanus gigas. (A–C, F, G) Maximum intensity projections. (D, E) 1 µm thick optical sections through the largest tooth of the gnathobase. (A, F) Distribution of resilin. (B) Chitinous exoskeleton (red) and resilin-dominated structures (blue). (C–E, G) Chitinous exoskeleton (red, orange), resilin-dominated structures (blue, light blue, turquoise) and silica-containing structures (green). Scale bars = 20 µm (A, B, C), 5 µm (D, E), 25 µm (F, G). (A–E) Adapted with permission from [29]. (F, G) Adapted with permission from [30], copyright 2015 Elsevier.

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ture with a large resilin proportion. On top, another cap-like mation in and breakage of the teeth, the soft and elastic resilin- structure that is composed of silica is located. All other gnatho- containing structures are supposed to function as flexible bear- base teeth are smaller, contain no silica, are mainly chitinous ings that can be compressed and thereby reduce stress concen- and have tips with large resilin proportions. C. hamatus is omni- trations in the tooth material and increase the resistance of the vorous and feeds on, among other organisms, diatoms and prot- teeth to mechanical damages. Additional structures with large ists. It is assumed that the large silica-containing teeth are used resilin proportions, located in the central and proximal parts of for feeding on diatoms. The silica makes these teeth stiffer and the gnathobases, are assumed to have a damping function that more mechanically stable and thereby more efficient in cracking makes the whole gnathobases resilient and further reduces the the diatom frustules. In case the diatom frustules are too stable risk of mechanical damage of the teeth. The smaller gnathobase and the pressure acting on the tips of the siliceous teeth exceeds teeth of C. hamatus are likely used to grab protists. In this the breaking stress level causing an increased risk of crack for- context, the grip of the tooth tips is suggested to be increased by

Figure 8: Material composition and properties of the ventral abdominal cuticle of females of the common bed bug (Cimex lectularius). (A) Abdomen overview (scanning electron micrograph). (B) Section of A indicating the locations of the spermalege (S) and three other cuticle areas called AS, M and AM, and penetration forces (mean ranks and standard errors) determined for these four cuticle sites. (C–L) Confocal laser scanning micrographs (maximum intensity projections) showing overlays of different autofluorescences exhibited by the exoskeletons. Blue colours indicate large proportions of resilin. (C, D) Autofluorescence composition of the cuticle in the left (C) and right (D) abdomen parts. The dominance of violet/blue autofluorescence (shown in blue) is restricted to the spermalege, clearly indicating that only at this site the cuticle contains large proportions of resilin. (E–H) Autofluorescence composition of the cuticle at the sites AS (E, F), M (G) and AM (H). The cuticle at M and AM consists mainly of sclerotised chitinous material, indicated by the dominance of autofluorescence shown in red, while the presence of large proportions of autofluorescence shown in green in the cuticle at AS indicates that the respective material consists mainly of weakly or non-sclerotised chitinous material. (I–L) Autofluorescence composition of the cuticle at the spermaleges of different one-week-old females, indicating variation of the extent of the resilin-dominated spermalege structures between females. Scale bars = 500 µm (A), 50 µm (C, D, F–L), 25 µm (E). Figure reproduced with permission from [126].

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the soft and elastic resilin making the grabbing process more Reproductive organs, mechanoreceptors and efficient. compound eyes The mating of bed bugs represents a famous example of sexual The gnathobases of the calanoid copepod Rhincalanus gigas, a conflict. During every successful mating event, the cuticle of species whose diet mainly consists of diatoms, are character- the ventral side of the abdomen of the female is penetrated by ised by five relatively large and compact teeth that possess a the male with a cannula-like intromittent organ, and the male combination of different materials comparable to that of the injects sperm and accessory gland fluids directly into the silica-containing teeth of C. hamatus (Figure 7F,G). Each of abdomen where the sperm migrate to the ovaries [123,124]. these teeth has a silica-containing cap-like structure, which is This traumatic insemination imposes survival costs on the located on a chitinous socket. At the base of the socket, the females [124] but the females cannot avoid mating [125]. As a gnathobase exoskeleton features large proportions of resilin. result of this sexual conflict, a female organ, the so-called sper- Like in the silica-containing teeth of C. hamatus, these resilin- malege, has evolved. In common bed bugs (Cimex lectularius), containing structures very likely function as compressible this organ is located on the right side of the ventral abdomen supports reducing the risk of mechanical damages of the teeth part where it is visible as a notch-like modification of the during feeding on diatoms with stable frustules. In general, the posterior edge of the fifth segment that exposes the subjacent complex composite systems in the gnathobase teeth are intersegmental membrane and cuticle of the sixth segment assumed to have co-evolved within an evolutionary arms race (Figure 8A,B). A recent study revealed that the spermalege together with the diatom frustules [122]. cuticle, by contrast to other cuticle sites analysed on the ventral

Figure 9: Resilin in mechanoreceptors. (A–F) Confocal laser scanning micrographs showing overlays of different autofluorescences exhibited by the exoskeletons. Blue colours indicate large proportions of resilin. (A) Dorsal view of the pretarsus of a third leg of a female drone fly (Eristalis tenax). (B) Dorsal view of a section of a second leg’s pretarsus of a male E. tenax. The arrow highlights a hair plate sensillum. (C) Larger view of the hair plate sensillum highlighted in B. (D, E) Confocal laser scanning micrographs showing 1 µm thick optical sections through the hair plate sensillum shown in C. (F) Cercal filiform hair (CFH) and associated campaniform sensilla (highlighted by small arrows) on a cercus of a female house cricket (Acheta domesticus). (A–C, F) Maximum intensity projections. Scale bars = 25 µm (A, B), 10 µm (C–F). Figure reproduced with permission from [10], copyright 2011 John Wiley and Sons.

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side of the female abdomen, contains large proportions of rather long and relatively thick hairs are present (Figure 9A–E). resilin [126] (Figure 8C–L). In microindentation tests, the The base of each hair is surrounded by a joint membrane that, penetration force necessary to pierce the resilin-rich sper- due to its resilin-dominated material composition, is soft and malege cuticle was significantly lower than that necessary to flexible and allows movement and bending of the hair shaft re- pierce the other cuticle sites [126] (Figure 8B). In addition, evi- sulting in a stimulation of the receptor. Because the long hairs dence for a significantly reduced tissue damage and hemo- project beyond and below the pulvilli of the pretarsus, they lymph loss was obtained for piercings of the spermalege cuticle touch the substrate shortly before the pulvilli and likely have the compared with piercings of the other cuticle sites [126]. The function to indicate the upcoming contact between the pulvilli results suggest that the material composition of the spermalege and the substrate. The cerci of crickets feature cercal filiform cuticle has evolved as a tolerance trait that reduces the mating hairs associated with campaniform sensilla [128,130]. The latter costs of both the female and the male: due to the softness of and the bases and sockets of the filiform hairs are embedded in resilin the penetration is easier for the male and causes less material that contains relatively large proportions of resilin [10] wounding of the female, and after the withdrawal of the intro- (Figure 9F) and, as mentioned above, very likely allow move- mittent organ the elasticity of resilin causes a sealing of the ment and bending of the shafts of the filiform hairs and the puncture reducing the hemolymph loss and the risk of bacterial campaniform sensilla, which are very sensitive strain receptors infection. (also called flex or displacement receptors), and thereby stimu- late the respective dendrite of the sensory cell. Hair plate sensilla and campaniform sensilla are typical mechanoreceptors that are common in insect exoskeletons [127- The presence of large proportions of resilin (in part also de- 129]. These receptors possess so-called joint membranes and scribed as ‘resilin-like protein’) in compound eye lenses has cap membranes that are composed of large proportions of been described for different arthropods [10,131-134] resilin [10,127,129]. On the dorsal side of the pretarsus of the (Figure 10). While other exoskeleton components are typically drone fly (Eristalis tenax), for example, hair plate sensilla with micro- and nano-structured, coloured and pigmented and, there-

Figure 10: Resilin in compound eyes. Confocal laser scanning micrographs (maximum intensity projections) depicting overlays of different autofluorescences exhibited by the exoskeleton. Blue structures contain large proportions of resilin. (A) Lateral view of the head of a male green lacewing (Chrysoperla carnea). (B) Frontal view of the head of a pharaoh ant (Monomorium pharaonis) worker. (C) Lateral view of the head of a beetle of the genus Circocerus. Scale bars = 100 µm (A, C), 50 µm (B). (A, B) Adapted with permission from [10], copyright 2011 John Wiley and Sons.

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fore, not suitable as material for optical elements, the pro- 12. Lyons, R. E.; Wong, D. C. C.; Kim, M.; Lekieffre, N.; Huson, M. G.; nounced transparency, the colourlessness and the amorphous- Vuocolo, T.; Merritt, D. J.; Nairn, K. M.; Dudek, D. M.; Colgrave, M. L.; Elvin, C. M. Insect Biochem. Mol. Biol. 2011, 41, 881–890. ness make resilin a perfect material for the construction of doi:10.1016/j.ibmb.2011.08.002 optical systems. 13. Elvin, C. M.; Carr, A. G.; Huson, M. G.; Maxwell, J. M.; Pearson, R. D.; Vuocolo, T.; Liyou, N. E.; Wong, D. C. C.; Conclusion Merritt, D. J.; Dixon, N. E. Nature 2005, 437, 999–1002. Exoskeleton structures with large proportions of resilin are doi:10.1038/nature04085 common among arthropods. This review demonstrates the broad 14. Rauscher, S.; Pomès, R. Structural Disorder and Protein Elasticity. In Fuzziness; Fuxreiter, M.; Tompa, P., Eds.; Advances in Experimental range of resilin functions in various exoskeleton structures. 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